DIRECT MICROSCOPIC AND

STANDARD PLATE COUNT OF

MICROORGANISMS IN MILK

DAVID YIN – 20298788

PARTNER: IKRAN ADEN

TA: BEY LING & ALEX RADEN

COURSE: BIOL 241L

LAB SECTION:  BIOL.241.104.1.LAB 

LAB TIME: TUESDAYS 7:00PM – 10:00 PM – B1 378

DATE OF EXPERIMENTS: TUESDAY, MAY 11, 2010 & TUESDAY, MAY 18, 2010
INTRODUCTION

Experimental Objective

The purpose of the experiment is to use the direct microscopic and standard plate count methods

to record evidence of bacteria growth on various milk mediums and quantifying the results, as

well as to identify how various milk bacteria grown in the environments when the milk is raw,

pasteurized, sterilized, or post-date.

Direct Microscopic Count

The direct microscopic count is a simple examination of the various microorganisms within the

desired sample viewed under a microscope. This involves taking a bacterial sample (in the case

of the experiment, samples of milk) and applying it onto a slide, where it can be magnified and

viewed using a standard microscope. This method is fast, simple, and does not require substantial

effort or equipment. The experimenter is able to see individual bacteria, which makes counting

individual bacteria easier, and also shows the morphology of the microorganism. However, there

needs to be a substantial population to be counted, or the results will be inaccurate. As well, it

may be tedious and tiring for an individual to scan and count every microorganism. Lastly, there

could be dead cells, or debris and air bubbles that look like cells; this would cause misleading

results.

Standard Plate Count

The standard plate count uses agar-plates to assist in growing bacteria and approximating

growth. This involves dilutions of a sample (in the case of the experiment, samples of milk) and

pour-plating onto or mixing into agar growth medium. After appropriate incubation, colonies
will form. The colonies are then counted – the goal is to find plates that have colony numbers

between 25-250 colonies. Under the assumption that a colony was formed by one bacterium and

using the dilution factor, one can calculate an estimated total number of bacteria on a specific

plate. This method is advantageous because it counts only viable bacteria (those that are alive).

In addition, experimenters are able to get more accurate results for samples with lower bacteria

populations. However, only the organisms that can thrive in the medium are grown, and so some

are unaccounted for. In addition, a colony can be formed by multiple cells, or could overlap

another colony, causing the data to be misleading. Lastly, as with the direct count method, “non-

cells” could be accidentally counted.

Microorganisms in Milk

Milk is a solution of various essential nutrients, like carbohydrates, proteins, and fats. Thus, milk

serves as an excellent growth medium for various microorganisms. Most of these are bacteria

belonging to either the bacillus or cocci family, such as Staphylococci, Micrococci,

Pseudomonas, Flavobacterium, and Erwinia. There could also be fungi present. These

organisms are usually introduced to the milk from the producer organism. Some specific

organisms, such as Streptococcus lactis, Acinetobacter johnsoni, and certain Lactobacilli can

cause milk to become slimey or sour. In more unsanitary conditions, disease-causing bacteria,

such as Salmonella, Mycobacterium bovis, and Staphylococcus aureus, could contaminate the

milk. Thus, pasteurization is necessary in order to make milk safe to drink.

Pasterization Methods

Pasterization is a process that removes harmful organisms from milk, thus lowering the overall

bacterial count. This is not the same as sterilization, which would kill all the microorganisms.
The common methods are High-Temperature Short Time (HTST) VS. Low Temperature Long

Time (LTLT). In HTST, milk is heated for at least 15 seconds at 71.6°C. In comparison, the

LTLT pasteurization heats milk for at least 30 minutes, but at 62.9°C. After pasteurization, milk

is usually chilled in refrigeration containers and ready to be shipped and/or packaged.

MATERIALS AND METHODS

All materials and methods follow the BIOL241L manual laboratory procedures*, and there were
no deviations from the norm.

EXPERIMENTAL RESULTS

A B C
(Raw Milk) (Fresh Milk) (Past Date Milk)
Cells/ml Cells/ml Cells/ml

2.6 x 107 0.0 2.2 x 106

6.7 x 106 3.9 x 106 1.7 x 107

4.1 x 106 3.6 x 106 8.9 x 106

6.6 x 107 0.0 8.1 x 106

1.1 x 107 3.2 x 105 2.0 x 107

7.4 x 107 1.8 x 107 4.2 x 106

6.2 x 107 8.0 x 106 7.4 x 106

A B C
(Raw Milk) (Fresh Milk) (Past Date Milk)
Cells/ml Cells/ml Cells/ml

6.7 x 106 3.9 x 106 1.7 x 107

 A B
(Raw Milk) (Fresh Milk)
CFU/ml CFU/ml

8.3 x 104 3.7 x 102

1.4 x 106 1.0 x 103

9.4 x 106 1.3 x 103

5.5 x 107 2.7 x 102

2.3 x 106 8.0 x 102

2.4 x 106 4.3 x 106

1.6 x 105 NA

6.3 x 106 5.2 x 103

2.6 x 106 3.0 x 102

7.0 x 104 4.0 x 103

1.9 x 105 NA

1.7 x 105 1.0 x 103

4.7 x 105 NA ?

7.3 x 103 NA ?

5.1 x 106 NA ?
 A B
(Raw Milk) (Fresh Milk)
CFU/ml CFU/ml

2.3 x 106 6.5 x 102

DISCUSSION

When comparing the results for the two experiments, it can be seen that, for both experiments,

the number of bacteria/colony forming units (CFU) within raw milk are higher than numbers

within fresh (pasteurized) milk. This makes logical sense, because the pasteurization process

would have killed most of the bacteria in the milk, thus yielding a substantially lower number.

For the direct count method, the number of bacteria in fresh milk is twice as few as the ones in

raw milk. However, with the standard plate count method, the number of bacteria in fresh milk is

more than 3500 times less than found in raw milk. In addition, the count for raw milk for both

experiments are relatively similar (both are numbers in the millions). Thus, one can conclude that

the standard plate count gives a more accurate presentation of the bacterial population. For the

direct count method, observation errors could have accounted for more bacteria perceived than

actually existing, which would increase numbers. In addition, the counted cells are all factored

with the microscope factor, and so numbers tend to be more dilated. It should be noted that, with
the standard plate count, only aerobic bacteria that could thrive in the medium were grown and

counted. It is possible that various organisms were unaccounted for, thus lowering the overall

count. Lastly, due to careful laboratory technique, debris and air bubbles were relatively non-

present, thus not effecting count results.

REFERENCES

APPENDIX I – RAW DATA AND CALCULATIONS